Amygdaloid kindling is anxiogenic but fails to alter object recognition or spatial working memory in rats.

Kindling in rats produces enduring behavioral changes that parallel the psychobehavioral disturbances frequently accompanying temporal lobe epilepsy. Some evidence suggests that the site of kindling is an important determinant of the type of behavioral changes observed following kindling, although this variable has not been systematically investigated. In the present experiments, the effects of amygdaloid kindling were assessed on a battery of behavioral tests we used previously to assess the effects of kindling in dorsal hippocampus or perirhinal cortex. Three generalized seizures were kindled with stimulation in or near the basolateral amygdala. One week later, rats were tested successively on measures of anxiety, activity, object recognition memory, and spatial working memory over a period of 3 weeks. Amygdaloid kindling produced increased anxiety, but spared all other behaviors assessed. This pattern of results is partially distinct from the previously described effects of perirhinal cortical kindling, which increases anxiety but also impairs object recognition memory, and is completely distinct from dorsal hippocampal kindling, which selectively increases activity and impairs spatial working memory. The observations suggest that kindling of distinct highly interconnected temporal lobe sites produces distinct patterns of behavioral comorbidity. The underlying mechanisms are thus most likely localized to intrinsic circuits at the site of seizure origination.

High-fat diet impairs hippocampal neurogenesis in male rats.

High fat diets and obesity pose serious health problems, such as type II diabetes and cardiovascular disease. Impaired cognitive function is also associated with high fat intake. In this study, we show that just 4 weeks of feeding a diet rich in fat ad libitum decreased hippocampal neurogenesis in male, but not female, rats. There was no obesity, but male rats fed a diet rich in fat exhibited elevated serum corticosterone levels compared with those fed standard rat chow. These data indicate that high dietary fat intake can disrupt hippocampal neurogenesis, probably through an increase in serum corticosterone levels, and that males are more susceptible than females.

Working memory training decreases hippocampal neurogenesis.

The relationship between adult hippocampal neurogenesis and cognition appears more complex than suggested by early reports. We aimed to determine if the duration and task demands of spatial memory training differentially affect hippocampal neurogenesis. Adult male rats were trained in the Morris water maze in a reference memory task for 4 days, or alternatively working memory for either 4 or 14 days. Four days of maze training did not impact neurogenesis regardless of whether reference or working memory paradigms were used. Interestingly, 2 weeks of working memory training using a hidden platform resulted in fewer newborn hippocampal neurons compared with controls that received either cue training or no maze exposure. Stress is a well-established negative regulator of hippocampal neurogenesis. We found that maze training in general, and a working memory task in particular, increased levels of circulating corticosterone after 4 days of training. Our study indicates that working memory training over a prolonged period of time reduces neurogenesis, and this reduction may partially be mediated by increased stress.

Platelet-derived growth factor (PDGF-BB) and brain-derived neurotrophic factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions.

The effects of i.c.v. infused platelet-derived growth factor and brain-derived neurotrophic factor on cell genesis, as assessed with bromodeoxyuridine (BrdU) incorporation, were studied in adult rats with unilateral 6-hydroxydopamine lesions. Both growth factors increased the numbers of newly formed cells in the striatum and substantia nigra to an equal extent following 10 days of treatment. At 3 weeks after termination of growth factor treatment, immunostaining of BrdU-labeled cells with the neuronal marker NeuN revealed a significant increase in newly generated neurons in the striatum. In correspondence, many doublecortin-labeled neuroblasts were also observed in the denervated striatum following growth factor treatment. Further evaluation suggested that a subset of these new neurons expresses the early marker for striatal neurons Pbx. However, no BrdU-positive cells were co-labeled with DARPP-32, a protein expressed by mature striatal projection neurons. Both in the striatum and in the substantia nigra there were no indications of any newly born cells differentiating into dopaminergic neurons following growth factor treatment, such that BrdU-labeled cells never co-expressed tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. In conclusion, our results suggest that administration of these growth factors is capable of recruiting new neurons into the striatum of hemiparkinsonian rats.

Anterior perirhinal cortex kindling produces long-lasting effects on anxiety and object recognition memory.

Temporal lobe epilepsy (TLE) is frequently accompanied by memory impairments and, although their bases are unknown, most research has focused on the hippocampus. The present study investigated the importance of another medial temporal lobe structure, the perirhinal cortex (Prh), in changes in memory in TLE using kindling as a model. Rats were kindled twice daily with anterior Prh stimulation until three fully generalized seizures were evoked. Beginning 7 days later and on successive days, rats were tested in an elevated plus maze, a large circular open field, an open field object exploration task and a delayed-match-to-place task in a water maze in order to assess anxiety-related and exploratory behaviour, object recognition memory and spatial cognition. Kindling increased anxiety-related behaviour in both the elevated plus and open field mazes and disrupted spontaneous object recognition but spared all other behaviours tested. These results are consistent with other findings indicating a greater role for the Prh in object memory and emotional behaviour than in spatial memory and contrast with the selective disruption of spatial memory produced by dorsal hippocampal kindling. The site-selectivity of the behavioural disruptions produced by kindling indicates that such effects are probably mediated by changes particular to the site of seizure initiation rather than to changes in the characteristic circuitry activated by limbic seizure generalization. Further investigation of the behavioural effects of Prh kindling may be useful for studying the mechanisms of mnemonic and affective dysfunction associated with TLE and offer insights into bases for variability in such dysfunction across patients.

The relation between extent of dorsal hippocampal kindling and delayed-match-to-place performance in the Morris water maze.

Dorsal hippocampal kindling impairs subsequent performance on spatial tasks. The relation between this effect and the extent of kindling achieved prior to testing has not been clearly established. Thus, the present study investigated the effects of dorsal hippocampal kindling on performance of a delayed-match-to-place (DMTP) task in the Morris water maze by assessing performance after each of series of different points in the kindling process including 1, 6, 11, and 16 afterdischarges, 1 stage 1 seizure, and 1 stage 5 seizure. We found that kindling produced a deficit that was apparent very early into kindling in terms of both direct swim (by 1 AD) and escape distance (by 6 ADs) measures but that did not clearly change in severity with further kindling. These results illustrate that kindling of even a few localized hippocampal seizures can disrupt spatial cognition and suggest that the mechanisms mediating memory disruption either do not change substantially as kindling progresses or that compensatory processes are engaged across training that mitigate any further kindling-related deteriorations in performance.

Caspase inhibitors increase short-term survival of progenitor-cell progeny in the adult rat dentate gyrus following status epilepticus.

The dentate gyrus (DG) is one of the few regions in the brain that continues to produce new neurons throughout adulthood. Seizures not only increase neurogenesis, but also lead to death of DG neurons. We investigated the relationship between cell death and neurogenesis following seizures in the DG of adult rats by blocking caspases, which are key components of apoptotic cell death. Multiple intracerebroventricular infusions of caspase inhibitors (pancaspase inhibitor zVADfmk, and caspase 3 and 9 inhibitor) prior to, just after, 1 day after, and 1 week following 2 h of lithium-pilocarpine-induced status epilepticus reduced the number of terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelled (TUNEL) cells and increased the number of bromodeoxyuridine (BrdU) -stained proliferated cells in the subgranular zone at 1 week. The caspase inhibitor-treated group did not differ from control at 2 days or 5 weeks following the epileptic insult. Our findings suggest that caspases modulate seizure-induced neurogenesis in the DG, probably by regulating apoptosis of newly born neurons, and that this action can be suppressed transiently by caspase inhibitors. Furthermore, although previous studies have indicated that increased neuronal death can trigger neurogenesis, we show here that reduction in apoptotic death may be associated with increased neurogenesis.

Dorsal hippocampal kindling produces a selective and enduring disruption of hippocampally mediated behavior.

Kindling produces enduring neural changes that are subsequently manifest in enhanced susceptibility to seizure-evoking stimuli and alterations in some types of behavior. The present study investigated the effects of dorsal hippocampal (dHPC) kindling on a variety of behaviors to clarify the nature of previously reported effects on spatial task performance. Rats were kindled twice daily with dHPC stimulation until three fully generalized seizures were evoked. Beginning 7 d later and on successive days, rats were tested in an elevated plus maze, a large circular open field, an open field object exploration task, and a delayed-match-to-place (DMTP) task in a water maze to assess anxiety-related and activity-related behavior (tasks 1 and 2), object recognition memory (task 3), and spatial cognition (task 4). Kindling disrupted performance on the DMTP task in a manner that was not delay dependent and produced a mild enhancement of activity-related behaviors in the open field task but not the elevated plus maze. All other aspects of testing were spared. These findings indicate that dHPC kindling produces enduring and selective effects on behavior that are consistent with a restricted disruption of hippocampally mediated functions. Possible bases for these effects are changes in local NMDA receptor function and/or changes in local inhibition, which might alter the optimal conditions for experience-dependent induction of intrahippocampal plasticity. This preparation may be useful for studying the mechanisms of mnemonic dysfunction associated with temporal lobe epilepsy and may offer unique insights into the mechanisms underlying normal hippocampal function.

Kindling of claustrum and insular cortex: comparison to perirhinal cortex in the rat.

The perirhinal cortex has recently been implicated in the kindling of limbic generalized seizures. The following experiments in rats tested the selectivity of the perirhinal cortex's epileptogenic properties by comparing its kindling profile with those of the adjacent insular cortex, posterior (dorsolateral) claustrum and amygdala. The first experiment examined the kindling and EEG profiles, and found that both the claustrum and insular cortex demonstrated rapid epileptogenic properties similar to the perirhinal cortex, including very rapid kindling rates and short latencies to convulsion. Furthermore, electrical stimulation of all three structures led to a two-phase progression through stage-5 seizures which had characteristics of both neocortical and amygdaloid kindling. In a second experiment rats were suspended in a harness to allow for more detailed documentation of both forelimb and hindlimb convulsions. With this procedure we were able to detect subtle yet unique differences in convulsion characteristics from each of the kindled sites and stage-5 seizure phases. Some of these convulsive parameters were correlated with changes in FosB/DeltaFosB protein and BDNF mRNA expression measured two hours after the last convulsion. Overall, it appears that the perirhinal cortex is not unique in its property of rapid epileptogenesis. Moreover, the posterior claustrum exhibited the fastest kindling and most vigorous patterns of clonus, suggesting that it may be even more intimately associated with the motor substrates responsible for limbic seizure generalization than is the perirhinal cortex.

Dorsal hippocampal kindling selectively impairs spatial learning/short-term memory.

Kindling with electrical stimulation of the dorsal hippocampus has been shown to disrupt spatial task performance in rats. The present study investigated the specificity of this effect in terms of the possible contribution of nonmnemonic effects, the presence of a more general mnemonic deficit, and the involvement of learning/short-term memory and/or long-term memory processes. Rats were fully kindled with stimulation of the dorsal hippocampus and subsequently tested for acquisition, 7-day retention, and 28-day retention of a hidden platform (HP) location in the Morris water maze and an object discrimination problem in a modified water maze. To control for nonmnemonic behavioral impairments, testing on both tasks was preceded by training on visible platform control tasks. Kindling impaired acquisition of the HP location but spared performance on all other aspects of testing, indicating a specific impairment of spatial learning/short-term memory. These results suggest that epileptogenesis induced by hippocampal stimulation is indeed associated with a selective disruption of the mechanisms mediating spatial learning/short-term memory.

Development and persistence of kindling epilepsy are impaired in mice lacking glial cell line-derived neurotrophic factor family receptor alpha 2.

Seizure activity regulates gene expression for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and their receptor components, the transmembrane c-Ret tyrosine kinase and the glycosylphosphatidylinositol-anchored GDNF family receptor (GFR) alpha 1 and alpha 2 in limbic structures. We demonstrate here that epileptogenesis, as assessed in the hippocampal kindling model, is markedly suppressed in mice lacking GFR alpha 2. Moreover, at 6 to 8 wk after having reached the epileptic state, the hyperexcitability is lower in GFR alpha 2 knock-out mice as compared with wild-type mice. These results provide evidence that signaling through GFR alpha 2 is involved in mechanisms regulating the development and persistence of kindling epilepsy. Our data suggest that GDNF and NRTN may modulate seizure susceptibility by altering the function of hilar neuropeptide Y-containing interneurons and entorhinal cortical afferents at dentate granule cell synapses.

Mossy fiber sprouting is dissociated from kindling of generalized seizures in the guinea-pig.

Controversy surrounds whether aberrant mossy fiber sprouting in the hippocampus is necessary for the establishment of seizure states. We investigated the association between mossy fiber sprouting and kindling in guinea-pigs, using either single-site or alternate-site stimulation. Kindling with single-site amygdaloid stimulation did not induce significant sprouting, despite the development of partial seizures. In contrast, single-site septal and alternating amygdaloid-septal stimulation produced moderate but significant sprouting in about 60% of animals that failed to develop stage 5 generalized seizures. Since the magnitude of sprouting was similar despite striking differences in the intensity of seizures that developed, we conclude that mossy sprouting is not causally associated with seizure development.

Claustral lesions delay amygdaloid kindling in the rat.

PURPOSE: Lesions of the claustrum in cats and primates have been shown to disrupt the development and expression of amygdaloid-kindled seizures in cats and primates. Because the structure and connectivity of the claustrum can vary between species, we wanted to examine the effects of claustral lesions on kindling in rats. METHODS: One group of rats received bilateral radiofrequency lesions of both anterior and posterior regions of the claustrum before amygdaloid kindling. Another group of rats received bilateral anterior and posterior radiofrequency lesions of the claustrum after amygdaloid kindling. Some rats were tested for transfer of kindling to the contralateral amygdala after claustral lesions. RESULTS: Small lesions that destroyed 13% of the claustrum were capable of delaying, but not blocking, amygdaloid kindling. The delay in kindling was due to an increase in the stimulation trials required to kindle to stage 5 seizures. The lesions had no effect on established kindled seizures or on transfer to the contralateral amygdala. CONCLUSIONS: As in other species, the claustrum in the rat appears to play a role in kindling from the amygdala. Because of the restricted size of our claustral lesions, however, we were unable to conclusively assess the full extent of the claustrum's participation in limbic kindling.

MK-801-induced expression of Fos protein family members in the rat retrosplenial granular cortex.

The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 has been shown to induce an acute Fos and Fos-related antigen (Fra) expression in the rat retrosplenial granular cortex (RSG), but the exact composition of the Fos protein family and their individual dynamic alterations are unknown. We examined this issue using immunocytochemistry and Western blot analysis with two antibodies that recognize, respectively, Fos and all the identified members of Fos protein family. Immunocytochemistry detected a rapid and transient expression of Fos proteins in some RSG neurons and a delayed and prolonged expression of Fra proteins in most RSG neurons following a single systemic MK-801 injection (1 mg/kg). Multiple MK-801 injections (i.e., ten consecutive injections once every other day; 1 mg/kg) produced a moderate Fra expression but failed to induce detectable Fos expression. Western blot analysis further showed a transient expression of 72-kDa Fos proteins following a single MK-801 injection and a prolonged expression of 46- and 43-kDa Fra proteins after either a single or multiple MK-801 administration. The delayed onset and prolonged expression of these Fra proteins suggest that they may be DeltaFosB. The possible relevance of these results to clinical psychotomimetic effects of the NMDA receptor antagonists phencyclidine and ketamine is discussed.

Dissociation between mossy fiber sprouting and rapid kindling with low-frequency stimulation of the amygdala.

In an attempt to determine whether sprouting of mossy fibers is invariably correlated with kindling of seizures, we subjected rats to rapid kindling with long trains of low-frequency stimulation of the amygdala that resulted in development of generalized seizures within a mean of five stimulations. For comparison, we subjected other rats to conventional kindling with short trains of high-frequency stimulation of the amygdala that resulted in development of generalized seizures within a mean of 13 stimulations. We found no evidence of mossy fiber sprouting in the dentate gyrus of rats killed one day after completion of rapid kindling, as compared to yoked controls, although significant sprouting was seen in rats killed one day after completion of conventional kindling. When we examined tissue from rats killed 20 days after rapid kindling, however, we did find significant sprouting, suggesting that mossy fiber sprouting can be triggered by rapid kindling if sufficient survival time is allowed. The observed disparity between completion of rapid low-frequency kindling and detection of mossy fiber sprouting suggests that mossy fiber sprouting may be associated more with sustained survival time after neuronal activation than with kindling per se. Furthermore, the similar time course of conventional kindling and of mossy fiber sprouting obscures the determination of a causal role of mossy fiber sprouting in conventional kindling.

Amygdala kindling-resistant (SLOW) or -prone (FAST) rat strains show differential fear responses.

The authors compared two rat strains, selectively bred for their susceptibility to amygdala kindling, with respect to their performance on various behavioral and learning tasks that are associated with fear and anxiety. The two rat strains differed significantly in measurements of exploration of novel and familiar environments, as well as in reactivity to footshock and fear-based learning. The kindling-resistant (SLOW) strain exhibited a lower ratio of open- to closed-arm entries in the elevated plus-maze, less activity over days in the open field, greater behavioral suppression in the open-field if previously footshocked, greater freezing in the inhibitory avoidance task, and slower acquisition and poorer retention in the one-way avoidance task than did the kindling-prone (FAST) strain. These experiments suggest that the SLOW rats are more expressively fearful than the FAST rats, particularly with respect to environmentally triggered freezing or immobility. Further, these observations imply that the relatively constrained excitability of the amygdala network in the SLOW rats might mediate their relatively greater expression of fear and anxiety compared with the FAST rats.

Time course of mossy fiber sprouting following bilateral transection of the fimbria/fornix.

Lesions of the fimbria/fornix (FF) lead to a variety of epileptiform changes in hippocampal activity. In epilepsy, as well as in several experimental preparations of epilepsy, aberrant sprouting of the dentate gyrus mossy fibers (MF) is observed and has been hypothesized to play a critical role in the generation of seizure activity. We therefore sought to determine whether MF sprouting also follows FF transections. FF transections did indeed lead to MF sprouting, which became apparent at 14 days and reached asymptotic levels at 28 days post-lesion. These results indicate a possible mechanism for the epileptiform activity seen following FF lesions and provide an additional example of the diverse epileptogenic treatments that are accompanied by MF sprouting.

The effects of fimbria/fornix transections on perforant path kindling and mossy fiber sprouting.

Various clinical and experimental studies of epilepsy have described synaptic reorganization in the dentate gyrus of hippocampus, in the form of collateral sprouting of the mossy fibers. These reports have led to the hypothesis that reorganized mossy fibers form a functional excitatory feedback circuit that contributes to local circuit hyperexcitability and chronic seizures. Much of the evidence supporting the sprouting hypothesis has been derived from kindling. We recently reported that transection of the fimbria/fornix (FF), which produces chronic epileptiform activity in the hippocampus, also induces mossy fiber sprouting in the inner molecular layer of the dentate gyrus. In the present study, we attempted to determine whether mossy fiber sprouting contributes to epileptiform activity, by examining the effects FF transections on perforant path (PP) kindling and associated mossy fiber sprouting. We found that FF transections and PP kindling produced moderate levels of sprouting, whereas the combination of the two treatments produced significantly denser sprouting. FF transections had mixed effects on kindling: afterdischarge thresholds were decreased and clonus and afterdischarge durations were increased, suggesting increased local excitation, whereas the kindling of behavioral seizures was delayed, suggesting decreased epileptogenesis.

Kindling antagonism: interactions of the amygdala with the piriform, perirhinal, and insular cortices.

We employed the kindling antagonism paradigm to characterize the role of the amygdala (AM), piriform cortex (PIR), perirhinal cortex (PRH), and insular cortex (INS) in the propagation and expression of generalized seizures. Alternating kindling stimulation was delivered to the AM and either the PIR, PRH, or INS. We found that kindling from the AM was retarded by stimulation only of the deep layers of both the INS and anterior PRH. Kindling antagonism was specific to the anterior cortical regions, since neither the posterior PRH or PIR were capable of antagonizing AM kindling. The results strongly implicate the deep layers of anterior limbic cortex in establishment of antagonism of AM kindling.

Differential sensitivity of various temporal lobe structures in the rat to kindling and status epilepticus induction.

Using focal brain stimulation (kindling), discrete seizures can be triggered from many neuroanatomic sites with varying degrees of facility. From several of these sites, protracted seizures or status epilepticus (SE) also can be triggered. To date, no comparison has been made between different brain sites in their sensitivity both to kindling and to SE development. In this report, we have compared the kindling profiles of three amygdala nuclei, namely the basal (BL), central (CE), and medial (ME) nuclei, to the adjacent piriform (PIR) and perirhinal (PRH) cortices. In addition, three weeks following kindling, the susceptibility of each kindled site to status epilepticus (SE) was assessed by exposing the site to 60 min of electrical stimulation. We observed that (a) during the course of daily kindling, the afterdischarge threshold dropped progressively and significantly in all structures, (b) the rate of kindling in the PRH and PIR cortices and the CE amygdala was significantly faster than either the BL or ME amygdala, (c) when discrete convulsions were triggered, the latency to forelimb clonus in the PRH cortex and CE amygdala was significantly shorter than the other three structures, and (d) despite being slower to kindle than most other sites, stimulation of the BL nucleus most readily triggered SE. The kindling data suggest that discharges triggered from the PRH and CE more readily access motor systems supporting limbic convulsions than discharges triggered from the BL, ME nuclei or the PIR cortex. On the other hand, the SE data indicate that the mechanisms and circuits associated with the development of discrete kindled seizures are not identical to those associated with the induction of limbic SE.